ESMO E-Learning Diagnostic Work Up in NSCLC and the … · 2019-08-23 · Zheng D, et al....

DIAGNOSTIC WORK UP IN NSCLC AND THE IMPORTANCE OF OPTIMAL TISSUE MANAGEMENT IN THE ERA OF PRECISION MEDICINE

Noemí Reguart, MD, PhD

Hospital Clinic Barcelona, Barcelona, Spain

Cristina Teixidó, MSc, PhD

Hospital Clinic Barcelona, Barcelona, Spain

OUTLINE

◆ Advanced NSCLC: a need for personalised therapy

◆ Diagnostic workup of lung cancer:

⁃ Type of samples

⁃ Morphology and histology

⁃ Material for biomarker studies

◆ Overview of key genetic alterations in NSCLC

⁃ Techniques for diagnose

◆ Guidelines for genetic testing

◆ Summary / conclusions

EVOLUTION OF LUNG CANCER DIAGNOSIS

Lung cancer classification has moved from histologic to molecular classification

H&E Molecular Testing

IHC

FISH

by 2000 2019

Genetic Testing Assays

FISH, fluorescent in situ hybridisation; H&E, hematoxylin and eosin stain; IHC, immunohistochemistry.

Images courtesy of Dr C Teixidó and Dr N Reguart

NOS, not otherwise specified. Adapted from Li T, et al. J Clin Oncol 2013;31:1039–49

NSCLC MOLECULAR CLASSIFICATION

NSCLC classification has moved from histologic to molecular subtyping

Molecular Testing: Adenocarcinoma and NSCLC-NOS histologies

DRIVER ONCOGENES IN LUNG ADENOCARCINOMA

BY ETHNICITY

USA/Europe East Asia

EGFR 5-15% 40-55%

KRAS 20-30% 8-10%

ALK 3-6% 3-5%

ROS1 1-2% 2-3%

BRAF 2-3% 0.5-1%

RET 1-2% 1-2%

MET 4% 1.3%

HER2 2-3% 2-3%

NTRK 1% <1%

More than 50% of all lung adenocarcinomas harbour driver oncogenes

Incidence of genomic driver is variable among ethnic populations

Kohno T, et al. Transl Lung Cancer Res 2015;4(2):156–64; Sehgal K, et al. Transl Cancer Res 2018; 7(Suppl 7):S779–S786;

Zheng D, et al. Oncotarget 2016;7(27):41691–702.

METASTATIC NSCLC: ESMO CLINICAL PRACTICE GUIDELINES

Stage IV Non-Squamous Cell Carcinoma

Molecular Tests Negative (PD-L1 expression)

Stage IV Squamous Cell Carcinoma

Molecular Tests Positive (ALK/BRAF/EGFR/ROS1)A BA B

LoE Levels of evidence (I-V); GoR nd grades of recommendation (A-E); TMB, tumour mutational burden.Planchard D, et al. Ann Oncol 2018;29(Supplement_4):iv192–iv237, by permission of Oxford University Press on behalf of the European Society for Medical Oncology.

OUTLINE



⁃ Type of samples







Interventional

radiologist,

pulmonologist or

surgeon obtains

tissue for

diagnosis Lab or

pathologist

sends the

report to the

clinical care

team

Results within 10 working days (receipt of sample

by the lab to the clinical care team)

Oncologist

receives

results

SAMPLE JOURNEY FOR TESTING

Diagnose

and subtype

◆ Before starting, review relevant clinical information,

such as:

- Sample site

- Clinical suspicion on primary lung tumour or metastasis

- Reason for biopsy: diagnosis, molecular evaluation,

resistance

- Smoking history

◆ Then, stablish diagnostic work up:

⁃ FIRST→ Establish malignancy

⁃ SECOND → Define type of malignancy

⁃ THIRD→ Perform molecular testing as appropriate and

prioritise testing according to individual patients

1 2 3

Personalised

treatment

Sample

pre-analytics

Perform

Molecular testing

Adapted from Aisner DL, et al. Am J Clin Pathol. 2012;138:332–46 Adapted from Thunnissen E, et al. Lung Cancer 2012;76(1):1–18

NSCLC, non-small cell lung cancerImages courtesy of Dr C Teixidó and Dr N Reguart

THE HARSH REALITY OF TUMOUR SAMPLES

FOR DIAGNOSIS

Type of samples: cytology and formalin-fixed paraffin embedded (FFPE)

◆ Diagnostic material is generally scarce and it is essential to balance requirements for an accurate histologic diagnosis with the

need for molecular analyses

◆ When biopsy and cytology material from an individual patient are analysed by different professional (histopathologists and

cytologists), it is essential that they share information

◆ 40% NSCLC diagnosed by cytology

Ideal Sample SizeThe Reality Sample Size

1. IHC should not be performed unless necessary!!!!

2. In NSCLC-NOS, IHC reduces the number of

inconclusive diagnoses (rate <10%)

3. Limiting IHC between 2 and 4 markers seems

reasonable

HISTOLOGY: IMMUNOHISTOCHEMICAL MARKERS

Type of malignancy

TUMOUR TYPE IHC

Adenocarcinoma (ADC) CK7, TTF-1

Squamous cell carcinoma (SCC) P40 (P63, less specific)

Neuroendocrine (LCNEC, SCLC)CD56 (NSE, chromogranin,

synaptophysin)

◆ In NSCLC biopsy samples the following terminology

should be used:

⁃ ADC IHC not required if diagnostic morphology present

⁃ SCC IHC not required if diagnostic morphology present

⁃ Carcinomas lacking clear differentiation by morphology

and special stains are classified as NSCLC-NOS

⁃ NOS that stain with adenocarcinoma markers, classified

as NSCLC, favor adenocarcinoma

⁃ NOS that stain with squamous markers, classified as

NSCLC, favor squamous cell carcinoma

ADC, adenocarcinoma; LCNEC, large cell neuroendocrine carcinoma; NOS not otherwise specified; NSCLC, non-small cell lung cancer; SCC, squamous cell carcinoma;

SCLC, small-cell lung cancer.Travis WD, et al. WHO 4th ed. Lyon, France: IARC Press; 2015

HISTOLOGY: IMMUNOHISTOCHEMICAL MARKERS

Type of malignancy

Squamous cell carcinoma, P40 positiveLung adenocarcinoma, TTF1 positive

Images courtesy of Dr D. Martinez, Hospital Clinic Barcelona

TISSUE QUALITY CONTROL FOR MOLECULAR TESTING

Pathologist review H&E section and mark areas for analysis

The molecular specialist must evaluate whether the percentage of tumour in a given specimen reaches the detection

threshold of the specific molecular test or tests

H&E

SAMPLE

T

T

T

Assess tumour percentage and viability

Select ADC if mixed for testing

ADC, adenocarcinoma; H&E, haematoxylin and eosin stain; T, tumour. Images courtesy of Dr C Teixidó and Dr N Reguart

MOLECULAR TECHNIQUES AVAILABLE IN

ROUTINE DIAGNOSIS

◆ IHC

Single-marker molecular tests Multiplex-marker molecular tests

Illumina NextSeq Qiagen GeneReaderIon Torrent S5Ion Torrent PGMIllumina MiSeq

◆ PCR ◆ FISH ◆ Multiplex test

◆ Massively parallel / NGS

FISH, fluorescent in situ hybridisation; IHC, immunohistochemistry; NGS, next generation sequencing; PCR, polymerase chain reaction. Images courtesy of Dr C Teixidó and Dr N Reguart

MOLECULAR TECHNIQUES AVAILABLE IN

ROUTINE DIAGNOSIS

Tissue requirements

H&E

SAMPLE

◆ IHC

◆ FISH

⌁50-100 cells*

Selection on tumour areas

⌁ 600-1000 cells*

◆ RT-PCR

◆ nCounter

◆ NGS

Whole tissue sections

*Numbers are approximations and tissue requirements depends on technique/ reagent/ platform

FISH, fluorescent in situ hybridisation; H&E, hematoxylin and eosin stain; IHC, immunohistochemistry; NGS, next generation sequencing;

RT-PCR, reverse transcription polymerase chain reactionImages courtesy of Dr C Teixidó and Dr N Reguart

STRATEGY TO MAXIMISE TISSUE FOR MOLECULAR TESTING

Molecular testing sections can be prepared:

Together with first H&E section

After initial H&E for histological diagnosis

H&E for histological diagnosis

Repeat

sectioning

For IHC

For Molecular Testing

H&E

◆ At the time of initial sectioning for morphologic diagnosis,

additional sections may be taken, in anticipation of IHC or

predictive markers analyses

⁃ Technicians should preserve as much tissue as

possible for further evaluations

◆ Using cytology samples, cell block preparation from clot

formed by remaining fluid should be obtained in

combination with smears to improve diagnostic information

H&E, haematoxylin and eosin stain; IHC, immunohistochemistryImages courtesy of Dr C Teixidó and Dr N Reguart

OUTLINE


◆ Diagnostic Workup of lung cancer:

⁃ Type of samples

⁃ Morphology and Histology

⁃ Material for Biomarker Studies





EGFREpidermal Growth Factor Receptor

HOTSPOTS FOR EGFR GENE MUTATIONS

◆ Activating (and sensitising) EGFR mutations

are predictive for response to EGFR tyrosine

kinase inhibitors

◆ More common:

⁃ Never-smokers

⁃ Women

⁃ Adenocarcinoma subtype

◆ Alterations:

⁃ Insertions

⁃ Deletions

⁃ Point mutations

Reprinted by permission Springer]: Mod Pathol, Molecular pathology of lung cancer: key to personalized medicine, Cheng L, et al. Copyright 2012

EGFR MUTATION ASSAYS

Adapted from IASLC Textbook in Thoracic Oncology 2014

METHODTUMOUR DNA

REQUIRED (%)

Sanger direct sequencing 25

Real time/ TaqMan PCR 10

High Resolution melting analysis 5-10

Cobas 5-10

Pyrosequencing 5-10

SNaPshot PCR 1-10

MALDI-TOF MS-based genotyping 5

Cycleave PCR 5

Fragment lenght and RFLP analysis 5

Allelic specific PCR/ Scorpion ARMS 1

MassARRAY 1

PNA- LNA PCR clamp 1

Denaturing HPLC 1

Massively parallel/ NGS 0.1

Sen

siti

vity

AMP, Association for Molecular Pathology; ARMS, amplification refractory mutation system; CAP, College of American Pathologists; HPLC, high performance liquid chromatography; IASLC, International

Association for the Study of Lung Cancer; LNA, locked nucleic acid; NGS, next generation sequencing; PCR, polymerase chain reaction; PNA, peptide nucleic acid; RFLP, restriction fragment length polymorphism

CAP/IASLC/AMP recommended assays for EGFR

genotyping testing

PCR-based

methods

Non-squamous-cell NSCLC

Adenocarcinoma

Large-cell carcinoma

Other

Targeted

NGS

Lindeman N, et al. Arch Pathol Lab Med 2018;142(3):321–46

cfDNA, cell-free DNA; TKI, tyrosine-kinase inhibitorLindeman N, et al. Arch Pathol Lab Med 2018;142(3):321-346;

Planchard D, et al. Ann Oncol 2018;29(Supplement_4):iv192-iv237, by permission of Oxford University Press on behalf of the European Society for Medical Oncology

EGFR TESTING IN CELL-FREE PLASMA DNA

The dynamic nature of EGFR resistance mechanisms can be monitored in cfDNA

◆ Insufficient evidence to support the use of cfDNA for

the diagnosis of primary lung adenocarcinoma

◆ When tissue is limited and/or insufficient for molecular

testing, physicians may use a cfDNA assay to identify

EGFR mutations as an alternative

◆ Physicians may use cfDNA methods to identify EGFR

T790M mutations in lung adenocarcinoma patients

with progression or secondary clinical resistance to

EGFR-targeted TKI

◆ Testing of the tumour sample is recommended if cfDNA

result is negative

ALKAnaplastic Lymphoma Kinase

NOS, not otherwise specified Soda M, et al. Nature 2007;448:561–66; Takeuchi K, et al. Clin Cancer Res 2009;15:3143–9; Zhang X, et al. Mol Cancer 2010;9:188;

Rodig SJ, et al. Clin Cancer Res 2009;15:5216–23; Shaw AT, et al. J Clin Oncol 2009;27:4247–53

ALK FUSION GENE

◆ ALK fusion gene results in formation of cytoplasmic

chimeric proteins with constitutive kinase activity

◆ The ALK fusion gene appears to be a distinct NSCLC

molecular subset susceptible to targeted inhibition

◆ Tends to be mutually exclusive with EGFR and

KRAS mutations

◆ More common:

⁃ Never or light smokers

⁃ Younger age

⁃ Adenocarcinoma subtype Pfizer, data on file

ALK FUSION GENE

Exon A20

Variants

of the -fusion protein

ALKEML4

ALKEML4

ALKEML4

ALKEML4

ALKEML4

ALKEML4

ALKEML4

ALKEML4

ALKKIF5B

ALKTFG

ALKKLC1

ALKPTPN3

ALKSTRN

Fusion protein Variant

Frequency

in ALK+

NSCLC

E13;A20 (V1) 33%

E6a/b;A20 (V3a/b) 29%

E20;A20 (V2) 9%

E14;A20 (V4) 3%

E18;A20 (V5) 2%

E14;A20 (V7) 2%

E2a/b;A20 (V5a/b) 2%

E17;A20 (V8) 1%

0.5%

Unknown %

Most common partner EML4 (90%)

Breakpoint within EML4 differ: 21 variants

EML4

136 14 15 2017 182Breakpoints

Breakpoints within EML4

Breakpoint within ALK always at exon 20

Produces oncogenic EML4-ALK-fusion protein

Protein activation by partner-provided

coiled-coil dimerisation domain

Gridelli C, et al. Cancer Treat Rev 2014;40(2):300–6; D‘Arcangelo M, et al. Curr Opin Oncol 2013;25(2):121–9; Hallberg B, et al. Nat Rev Cancer 2013;13(10):685-700;

Ou SI, et al. Oncologist 2012;893:179–87.

FFPE, formalin-fixed paraffin embedded; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistryLindeman N, et al. Arch Pathol Lab Med 2018;142(3):321–46

TESTING FOR ALK GENE FUSION

ALK fusion variants in NSCLC

TECHNIQUES

Fluorescent in-situ hybridisation (FISH)

Immunohistochemistry (IHC)

Reverse transcription polymerase chain reaction (RT-PCR)

nCounter

Next Generation Sequencing (NGS)

◆ The ALK break-apart FISH assay detects ALK fusions in FFPE NSCLC tissue specimens

◆ Considered ALK positive if >15% cell with break-apart

◆ The centromeric (green) and telomeric (red) probes flank the ALK locus

⁃ Splitting probes of the red and green signals indicates ALK fusion

⁃ A yellow signal indicates no ALK fusion

◆ ALK IHC (D5F3, Ventana or 5A4, Novocastra) may be used as a screening test

FFPE, formalin-fixed paraffin embedded; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistryLindeman N, et al. Arch Pathol Lab Med 2018;142(3):321–46


Diagnostic algorithm that uses IHC as the primary test for ALK identification

ALK IHC

Negative

Score 0/1+Equivocal

Score 2+Positive

Score 3+

Reported as ALK NEGATIVE

ALK FISH

PositiveNegative

Reported as ALK POSITIVE

◆ Considered ALK IHC positive if strong granular

cytoplasmic staining with/without membrane

accentuation

◆ Occasional cases may be difficult to interpret

because of heterogeneous fixation/preservation

and/or nonspecific staining artefacts (e.g.,

staining in alveolar macrophages, neural cells,

extracellular mucin, necrosis). In these settings,

these cases should also be tested by a

validated method (e.g., ALK FISH)


IHC 5A4 NovocastraIHC D5F3 VentanaFISH BAP Menarini

Split

signal

Non-split

signal

Non-split

signal

BAP, break-apart probe; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistry Images courtesy of Dr C Teixidó and Dr N Reguart

Fernandez-Martínez A, et al. Cancer Treatment Communications 2016;6:4–7

ROS1c-ROS Oncogene 1

◆ The ROS1 fusion gene appears to be a distinct NSCLC


◆ Tends to be mutually exclusive with EGFR, KRAS,

ALK alterations

◆ More common:


⁃ Younger age


◆ The mechanism by which the ROS1 fusion protein is

activated remains unclear

Bergethon K, et al. J Clin Oncol 2012;30(8):863–70; Shaw AT, et al. N Engl J Med 2014;371(21):1963–71;

Rossi G, et al. Lung Cancer Targets and Therapy 2017:8 45–55. Images available under Non Commercial (unported, v3.0) License. Available at: http://creativecommons.org/licenses/by-nc/3.0/. Accessed Jun 2019.

ROS1 FUSION GENE

ROS1 FUSION GENE

Many partners, most common CD74 (30%)

ROS1

32 3534Breakpoints

Breakpoints within ROS1

Breakpoint sites vary exons 32, 34, 35

Produces oncogenic ROS1 kinase protein

ROS1Partner

Unknown mechanism of protein activation (no coiled-coil

dimerisation domain provided)

KinaseDimerisation domain

Shaw AT, et al. N Engl J Med 2014;371(21):1963–71; Takeuchi K, et al. Nat Med 2012;18(3):378–81;

Republished with permission of Pioneer Bioscience Publishing Company, from Transl Lung Cancer Res, Kohno T, et al. 4(2), 2015; permission conveyed through Copyright Clearance Center, Inc.

FISH, fluorescent in situ hybridisation; IHC, immunohistochemistryLindeman N, et al. Arch Pathol Lab Med 2018;142(3):321–46

TESTING FOR ROS1 GENE FUSION

TECHNIQUES



Reverse transcription polymerase chain

reaction (RT-PCR)

nCounter


◆ FISH is the trial-validated standard. The ROS1 FISH

testing should be performed with a break-apart probe

design given the multiple fusion partners described

◆ Considered ROS1 positive if >15% cell with break-apart

◆ ROS1 IHC (D4D6, Cell Signalling Technology) may be

used as a screening test in lung adenocarcinoma

patients; however, positive ROS1 IHC results should be

confirmed by an orthogonal method - cytogenetic method

(FISH) or molecular

BRAFB-raf Proto-Oncogene

◆ Activating mutations in BRAF, especially p.V600E, lead to

oncogenic signaling through MAPK

◆ BRAF V600E mutations appear to be a distinct NSCLC

molecular subset susceptible to targeted inhibition:

BRAF/MEK inhibitors

◆ Typically mutually exclusive of other oncogenic drivers

◆ It is appropriate to include BRAF as either part of larger

testing panels performed initially or when routine EGFR,

ALK, and ROS1 testing are negative

HOTSPOTS FOR BRAF GENE MUTATIONS

Lindeman N et al. Arch Pathol Lab Med 2018; 142(3):321-346

Planchard D et al. Ann Oncol 2018 1;29(Supplement_4):iv192-iv237From files of Teixidó C, Reguart N

N, N-terminal; C, C-terminal; RBD, RAS-binding domain; PL, P-loop; αC, alpha C hélix motif ; DF, dimerization interface; CL, catalytic loop; AS, activation site; aa, amino acid

CN

BRAF Kinase Domain

ASRBD PL ⍺C DF CL

717 766aa0 457

K601G596

V600

D594N581G466

G464

G469

DFG

RETREarranged during Transfection

RET FUSION GENE

Not currently indicated as routine stand-alone test outside the context of a clinical trial

◆ The RET fusion gene appears to be a distinct NSCLC


◆ Tends to be mutually exclusive with other oncogenic

drivers

◆ More common:


⁃ Younger age


◆ To date, no RET-directed targeted therapeutic has

received regulatory approval for RET-mutant or RET-

rearranged solid tumours

Ju YS, et al Genome Res 2012;22:436–45;

Ferrara R, et al. J Thorac Oncol. 2018;13(1):27–45. Image available under the terms of the Creative Commons Attribution-NonCommercial-No Derivatives License (CC BY NC ND) Available at

https://creativecommons.org/licenses/by-nc-nd/4.0/ Accessed June 2019.

RET FUSION GENE


Six partners, most common KIF5B (30%)

RET

1211Breakpoints

Breakpoints within RET

Breakpoint sites vary exons 11, 12

Produces oncogenic RET kinase protein

Protein activation by partner-provided

coiled-coil dimerisation domain

RETPartner

Takeuchi K, et al. Nat Med 2012; Figure republished with permission of Pioneer Bioscience Publishing Company, from Transl Lung Cancer Res, Kohno T, et al. 4(2), 2015;

permission conveyed through Copyright Clearance Center, Inc.

TESTING FOR RET GENE FUSION

TECHNIQUES



reaction (RT-PCR)

nCounter


◆ The RET FISH testing should be performed with a break-apart probe design given the multiple fusion partners described

◆ Considered RET positive if >15% cell with break-apart

◆ RET fusion genes cannot be adequately detected by IHC


IHC, immunohistochemistry; FISH, fluorescent in situ hybridisationLindeman N, et al. Arch Pathol Lab Med 2018; 142(3):321-346; Ferrara R, et al. J Thorac Oncol 2018;13(1):27–45

Ferrara R, et al. J Thorac Oncol 2018;13(1):27–45. Available under NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Available at https://creativecommons.org/licenses/by-nc-nd/4.0/,

accessed June 2019.

NTRKNeurotrophic tyrosine kinase

NTRK FUSION GENE

Family of neurotrophin receptors (NTRK 1-3)

NTRK fusion gene appears to be a distinct NSCLC


◆ Tends to be mutually exclusive with other

oncogenic drivers

◆ Prevalence in unselected population

⁃ Occur across age and smoking status

⁃ No gender preference

◆ To date, no NTRK-directed targeted therapeutic

has an approval for NTRK-gene fusion

Farago AF, et al. J Thorac Oncol 2015;10(12):1670–4. Available under NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Available at https://creativecommons.org/licenses/by-nc-nd/4.0/,

accessed June 2019;

Farago AF, et al J Thorac Oncol 2015;10(12):1670–4; Farago AF, et al. JCO Precis Oncol 2018; DOI: 10.1200/PO.18.00037

NTRK FUSION GENE

FISH

FISH

NTRK1

1412Breakpoints

Breakpoints within NTRK1

Breakpoint sites vary exons 12, 14

Oncogenic constitutive activation of TRKA


Most common partners CD74 and MPRIP

Neurotrophic receptors: NTRK1, NTRK2, NTRK3

Transmembrane proteins: TRKA, TRKB and TRKC

Most common fusion in lung NTRK1

Unknown mechanism of protein activation

(no coiled-coil dimerisation domain provided)

NTRKPartner

Vaishnavi A, et al. Nat Med 2013;19(11):1469–72; David Hyman, 2017 ASCO Annual Meeting. By permission of Dr D. Hyman.

TESTING FOR NTRK GENE FUSION

TECHNIQUES

Fluorescent in-situ hybridization (FISH)



reaction (RT-PCR)

nCounter


◆ TRK protein testing can be considered as part of broad immunohistochemistry testing

◆ TRK IHC may be used as a screening test

◆ A TRK positive test should then be confirmed with NGS

NCCN Clinical Practice Guidelines in Oncology, Version 2.2019. Reprinted by permission from Springer Nature, Mod Pathol, Molecular characterization of cancers with NTRK gene fusions. Gatalica Z, et al. 32(1):147–53,

Copyright 2019.

IHC, immunohistochemistry; NGS, next generation sequencing

METMET Proto-Oncogene

TESTING FOR MET GENE AMPLIFICATION


◆ FISH is the trial-standard technique but there is no guideline for cut-off

of MET positivity

◆ High polysomy occurs when there are multiple copies of chromosome 7

(CEP7) in tumour cells (>5)

◆ MET amplification may be classified by using MET:CEP7 Ratio as low

(≥1.8 to 2.2≤), intermediate (>2.2 to <5), and high (≥5)

◆ Low–intermediate levels can occur synchronously with other oncogenic

mutations and gene rearrangements up to 63% of lung carcinomas

TECHNIQUES



Real time polymerase chain reaction (qRT-PCR)

nCounter


Noonan SA, et al. J Thorac Oncol 2016;11(12):2253–8; Drilon A, et al. J Thorac Oncol 2017;12(1):15–26

Image courtesy of Dr C Teixidó and Dr N Reguart

TESTING FOR MET EXON 14 SKIPPING MUTATIONS


Schematic illustration of some MET△14

◆ MET△14 mutations exhibit a highly diverse sequence composition

(insertions, deletions, SNV)

TECHNIQUES

In-situ hybridisation (ISH)

Direct Sequencing

Reverse transcription polymerase chain reaction

(RT-PCR)

nCounter


SNV, single nucleotide variationReprinted from Cancer Discov 2015, 5(8):842–9, Paik PK, et al. Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping, with permission from AACR.

PD-L1Programmed Cell Death Ligand-1

IHC, immunohistochemistryImages courtesy of Dr C Teixidó and Dr N Reguart

PD-L1 TESTING FOR IMMUNOTHERAPY

Standard Detection by IHC

◆ PD-L1 expression has been detected on tumour cells and

tumour-infiltrating immune cells

◆ PD-L1 on tumour cells may lead to the inhibition of

activated T-cells

◆ To enrich for those patients more likely to benefit from

anti-PD-1 or anti-PD-L1 therapy

◆ IHC to identify PD-L1 expression at the appropriate level

and on the appropriate cell population(s) as determined by

the intended drug and line of therapy

TPS <1%

TPS 1–49%

TPS ≥50%

PD-L1 IHC ASSAYS IN LUNG CANCER

Summary of PD-L1 antibodies and technical aspects for evaluation in NSCLC

mAb cloneAb host

speciesPlatform

PD-L1

scoringCut-offs

22C3 Mouse Dako TCTC ≥1% or

TC ≥50%

28-8 Rabbit Dako TC TC ≥1%

SP142 Rabbit Ventana TC, IC TC ≥50% or IC

≥10%

SP263 Rabbit Ventana TC TC ≥25%

73-10 Rabbit Dako TC TC ≥1%

Blueprint Phase 2a

22C3, 28-8 and SP263 assays are

comparable when used to determine

PD-L1 status of patient’s tumour (TPS),

SP142 detects less, while 73-10 stains

more PD-L1 positive tumour cells

TC, tumour cells; IC, immune cells; TPS, tumour proportion scoreAdapted from Teixidó C, et al. Ther Adv Med Oncol 2018;10:1–17

Tsao MS, et al. J Thorac Oncol. 2018 Sep;13(9):1302-1311

OUTLINE



⁃ Type of samples







1

ALK

2

ROS1

KRAS

RET HER2MET

NSCLC Recommendations

ESMO Guidelines

‘Must-test’

‘Should-test’

BRAFEGFR PD-L1

◆ Testing for EGFR mutation status [LoE, GoR: I, A],

ALK rearrangement [I, A], ROS1 rearrangement [II,

A], BRAF V600 mutation status [II, A] and PD-L1

expression by IHC [I, A] should be systematically

analysed in advanced NSCLC

◆ Molecular EGFR and ALK testing are not

recommended in patients with a confident

diagnosis of squamous, except in unusual cases,

eg, never/former light smokers or long-time

ex-smokers [IV, A]

METASTATIC NSCLC: ESMO CLINICAL PRACTICE

Molecular pathology/biology recommendations

LoE, levels of evidence (I–V); GoR, grades of recommendation (A–E); NSCLC, non-small cell lung cancer; IHC, immunohistochemistry; FISH, fluorescent in situ hybridisationPlanchard D, et al. Ann Oncol 2018;29(Supplement_4):iv192-iv237, by permission of Oxford University Press on behalf of the European Society for Medical Oncology.


Molecular pathology/biology recommendations

◆ EGFR mutation testing should have adequate coverage of mutations in exons 18–21, including those

associated with resistance to some therapies [LoE, GoR: III, B]

◆ Detection of the ALK translocation by FISH remains a standard, but IHC with high-performance ALK

antibodies and validated assays may be used for screening [III, A] and have recently been accepted as

an equivalent alternative to FISH for ALK testing

◆ Detection of the ROS1 translocation by FISH remains a standard; IHC may be used as a screening

approach [IV, A]

◆ If available, multiplex platforms (NGS) for molecular testing are preferable [III, A]. Whatever testing

modality is used, it is mandatory that adequate internal validation and quality control measures are in

place and that laboratories participate in, and perform adequately in, external quality assurance schemes

for each biomarker test [III, A]

FISH, fluorescent in situ hybridisation; GoR, grades of recommendation (A–E); IHC, immunohistochemistry; LoE, levels of evidence (I–V); NGS, next generation sequencing.Planchard D, et al. Ann Oncol 2018;29(Supplement_4):iv192-iv237


Biomarker testing

FISH, fluorescent in situ hybridisation; GoR, grades of recommendation (A–E); IHC, immunohistochemistry; LoE, levels of evidence (I–V); MEK, mitogen-activated protein kinase

kinase; NGS, next generation sequencing; TKI, tyrosine-kinase inhibitorPlanchard D, et al. Ann Oncol 2018;29(Supplement_4):iv192-iv237, by permission of Oxford University Press on behalf of the European Society for Medical Oncology.

SUMMARY / CONCLUSIONS

◆ In NSCLC, the determination of histologic subtype and molecular predictive markers are

standard of care

◆Determination of EGFR, ALK, ROS1, BRAF and PD-L1 status in tumour specimens is

currently standard of care in advanced NSCLC patients

◆ In lung cancer, optimal management of biopsy specimens are needed to avoid repeat biopsies

⁃Pathologists have a key role in treatment decisions

⁃Closed interaction between pulmonologists, pathologists, biologists, and oncologists is

required

◆ In the era of personalised therapy, professionals involved in lung cancer diagnosis/

management should develop their own multidisciplinary tissue management strategy to obtain

specimens and process them

THANK YOU!

Noemí Reguart [email protected]

Cristina Teixidó [email protected]

mailto:[email protected]

mailto:[email protected]

ESMO E-Learning Diagnostic Work Up in NSCLC and the … · 2019-08-23 · Zheng D, et al....

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